The present invention relates to a method for the quantitative determination of a specific component, e.g., 1,5-anhydroglucitol (hereinafter referred to as 1,5-AG) in a sample by utilizing an enzyme reaction. The method involves pretreatment which converts glucose in the sample into another substance. The invention also relates to a reagent and a reagent kit useful in the method.
Biological samples contain glucose, sometimes at very high concentrations compared with analytes, which may reduce the accuracy of assay results for analytes. In such cases, prior to the determination of analytes, glucose in samples is removed therefrom or converted into substances which do not interfere with the determination of analytes.
Previous methods for removing glucose from samples include methods in which glucose is separated by ion exchange column chromatography (Japanese Published Unexamined Patent Applications Nos. 185307/88 and 6756/89). Previous methods for converting glucose in samples into other substances include (1) a method in which glucose is converted into glucose-6-phosphate by a reaction utilizing the action of a phosphorylated enzyme such as glucokinase or hexokinase in the presence of adenosine triphosphate (ATP), and (2) a method in which glucose is converted into gluconolactone by a reaction utilizing the action of oxidase such as glucose oxidase, pyranose oxidase or sorbose oxidase in the presence of oxygen.
Further, various improvements have been made to these methods for the conversion of glucose. For example, modifications of the above method (1) using a phosphorylated enzyme include the following: a method in which glucose is converted into fructose-1,6-diphosphate by the action of phosphohexose isomerase and 6-phosphofructokinase in order to prevent the reconversion of glucose-6-phosphate into glucose by equilibrium reaction (Japanese Published Unexamined Patent Application No. 76397/93); methods using glucose-6-phosphate dehydrogenase in the presence of oxidized coenzymes (Japanese Published Unexamined Patent Applications Nos. 320998/89, 27299/91 and 237794/94); and a method using pyruvate kinase in the presence of adenosine diphosphate (ADP) to prevent the change in concentration of ATP, which decreases in the elimination of glucose, and to keep the ATP concentration constant (Japanese Published Unexamined Patent Application No. 104298/90). Modifications of the above method (2) using oxidase include a method in which a reaction using glucose oxidase is carried out and then the formed hydrogen peroxide is eliminated by the action of catalase (Japanese Published Unexamined Patent Application No. 185397/88).
However, the above methods suffer from the defect that enzyme reaction systems for the determination of analytes may be affected by the substances used for the conversion of glucose into other substances and the substances formed in the conversion system as well as their concentration. For example, when glucose is eliminated by the use of glucokinase or hexokinase (Japanese Published Unexamined Patent Application No. 76397/93), ADP is unfavorably formed in large quantities. Particularly, when a sufficient amount of ATP is supplied in order to completely eliminate glucose, ADP is formed at a concentration which is two times higher than that of glucose. The influence of ADP at such concentration on the reaction systems is not negligible.
1,5-AG is present in biological fluids such as cerebrospinal fluid, blood plasma, serum and urine. The level of 1,5-AG in blood plasma decreases in patients of certain diseases, especially diabetes, and thus 1,5-AG is useful as a diagnostic marker for diabetes. However, the determination of 1,5-AG is very difficult because of the close similarity in structure between 1,5-AG and glucose and the small quantity of 1,5-AG compared with glucose.
It is known that enzymes such as sorbose oxidase, pyranose oxidase, hexokinase, glucokinase and ADP-dependent hexokinase act on 1,5-AG, but these enzymes react also with sugars such as glucose which coexist with 1,5-AG. Therefore, some measures must be taken for removing or eliminating these sugars such as glucose.
Some methods comprising the step of elimination of glucose are known for the 1,5-AG determination in which 1,5-AG is oxidized using the catalytic action of pyranose oxidase or sorbose oxidase and the formed hydrogen peroxide is determined.
Examples of such methods are: (1) methods for 1,5-AG determination which comprise separating glucose in a sample by ion column chromatography, contacting the sample with pyranose oxidase, and determining the formed hydrogen peroxide (Japanese Published Unexamined Patent Applications Nos. 185307/88 and 6756/89); (2) methods for 1,5-AG determination which comprise converting glucose in a sample into a compound which does not react with pyranose oxidase by the action of glucokinase or hexokinase and glucose-6-phosphate dehydrogenase, contacting the sample with pyranose oxidase, and determining the formed hydrogen peroxide (Japanese Published Unexamined Patent Applications Nos. 320998/89 and 27299/91); (3) a method for 1,5-AG determination which comprises converting glucose in a sample into a compound which does not react with pyranose oxidase by the action of glucokinase and pyruvate kinase, contacting the sample with pyranose oxidase, and determining the formed hydrogen peroxide (Japanese Published Unexamined Patent Application No. 104298/90); and (4) a method for 1,5-AG determination which comprises converting glucose in a sample into a compound which does not react with pyranose oxidase by the action of hexokinase, phosphohexose isomerase and 6-phosphofructokinase, contacting the sample with sorbose oxidase or pyranose oxidase, and determining the formed hydrogen peroxide (Japanese Published Unexamined Patent Application No. 76397/93).
However, the above methods (1) using columns are disadvantageous because of their complicatedness in operation, and the above methods (2)-(4) have the disadvantage that hexokinase and glucokinase used in their glucose elimination systems act also on 1,5-AG to form 1,5-AG-6-phosphate, which reduces the accuracy of the assays for 1,5-AG.
Further, some methods comprising the step of elimination of glucose are known for the 1,5-AG determination in which 1,5-AG is phosphorylated using the catalytic action of hexokinase, glucokinase or ADP-dependent hexokinase and the formed substance is determined.
Examples of such methods are: (5) a method for 1,5-AG determination which comprises separating glucose in a sample by ion column chromatography, contacting the sample with hexokinase or glucokinase, and determining the formed ADP (Japanese Published Unexamined Patent Application No. 107796/96); and (6) a method for 1,5-AG determination which comprises converting glucose in a sample into a compound which does not react with ADP-dependent hexokinase by the action of (a) glucose oxidase, or glucose oxidase and catalase, (b) glucose dehydrogenase, or (c) hexokinase or glucokinase, contacting the sample with ADP-dependent hexokinase and 1,5-AG-6-phosphate dehydrogenase, and determining the formed reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] (Japanese Published Unexamined Patent Appllication No. 191998/98).
However, these methods suffer from the following disadvantages. Method (5) requires complicated operations. In method (6), in the case of (a) using glucose oxidase, oxygen supply is a rate-limiting step when glucose is present in a sample at a high concentration; in the case of (b) using glucose dehydrogenase, a system for eliminating NAD(P)H formed from glucose is required; and in the case of (c) using hexokinase or glucokinase, fractional determination can not be carried out because 1,5-AG-6-phosphate dehydrogenase acts on glucose-6-phosphate formed from glucose, and when the method is applied to samples containing a large quantity of glucose such as those from diabetes patients, ADP formed in the reaction system in a large quantity has an unfavorable effect on the accuracy of the assay for 1,5-AG.
Under the circumstances, an object of the present invention is to provide a simple method for the determination of a specific component, e.g. 1,5-AG, in a sample containing glucose, a method for substantially completely eliminating glucose in a sample in the determination of a specific component in the sample, and a reagent and a kit useful in the methods.
The present invention relates to a method for determining 1,5-AG in a sample containing glucose, which comprises contacting the sample with an enzyme system which converts glucose into fructose-1,6-diphosphate and converts 1,5-AG into 1,5-AG-6-phosphate to form 1,5-AG-6-phosphate, dehydrogenating 1,5-AG-6-phosphate in the sample by the action of 1,5-AG-6-phosphate dehydrogenase in the presence of an oxidized coenzyme, and measuring the amount of the reduced coenzyme formed by the dehydrogenation reaction.
The present invention also relates to a reagent for the determination of 1,5-AG, comprising (a) nucleosidediphosphate (hereinafter referred to as NDP), nucleoside triphosphate (hereinafter referred to as NTP), NDP-dependent hexokinase, phosphohexose isomerase, 6-phosphofructokinase, an oxidized coenzyme and 1,5-AG-6-phosphate dehydrogenase, or (b) one member selected from the group consisting of NTP-dependent hexokinase and NTP-dependent glucokinase, NTP, phosphohexose isomerase, 6-phosphofructokinase, an oxidized coenzyme and 1,5-AG-6-phosphate dehydrogenase.
The present invention further relates to a reagent kit for the determination of 1,5-AG, comprising (a) a reagent comprising NDP, NTP, NDP-dependent hexokinase, phosphohexose isomerase and 6-phosphofructokinase, or (b) a reagent comprising one member selected from the group consisting of NTP-dependent hexokinase and NTP-dependent glucokinase, NTP, phosphohexose isomerase and 6-phosphofructokinase, and a reagent comprising an oxidized coenzyme and 1,5-AG-6-phosphate dehydrogenase.
The present invention further relates to a method for eliminating glucose in a sample, which comprises converting glucose in the sample into fructose-1,6-diphosphate by the action of NDP-dependent hexokinase, phosphohexose isomerase and 6-phosphofructokinase in the presence of NDP and NTP.
The present invention further relates to a reagent for the elimination of glucose, comprising NDP, NTP, NDP-dependent hexokinase, phosphohexose isomerase and 6-phosphofructokinase.
The present invention further relates to a method for determining an analyte in a sample containing glucose, which comprises converting glucose in the sample into fructose-1,6-diphosphate by the action of NDP-dependent hexokinase, phosphohexose isomerase and 6-phosphofructokinase in the presence of NDP and NTP, and determining the analyte in the sample by using a chemical or enzymatic reaction.
The present invention further relates to a reagent for the determination of an analyte, comprising NDP, NTP, NDP-dependent hexokinase, phosphohexose isomerase, 6-phosphofructokinase, and an enzyme acting on the analyte or a substance reacting with the analyte.
The present invention further relates to a reagent kit for the determination of an analyte, comprising a reagent comprising NDP, NTP, NDP-dependent hexokinase, phosphohexose isomerase and 6-phosphofructokinase, and a reagent comprising an enzyme acting on the analyte or a substance reacting with the analyte.